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J Biol Chem, Vol. 274, Issue 46, 32680-32691, November 12, 1999

Ruthenium Red Modifies the Cardiac and Skeletal Muscle Ca²⁺ Release Channels (Ryanodine Receptors) by Multiple Mechanisms

From the Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599-7260

The effects of ruthenium red (RR) on the skeletal and cardiac muscle ryanodine receptors (RyRs) were studied in vesicle-Ca²⁺ flux, [³H]ryanodine binding, and single channel measurements. In vesicle-Ca²⁺ flux measurements, RR was more effective in inhibiting RyRs at 0.2 µM than 20 µM free Ca²⁺. [³H]Ryanodine binding measurements suggested noncompetitive interactions between RR inhibition and Ca²⁺ regulatory sites of RyRs. In symmetric 0.25 M KCl with 10-20 µM cytosolic Ca²⁺, cytosolic RR decreased single channel activities at positive and negative holding potentials. In close to fully activated skeletal (20 µM Ca²⁺ + 2 mM ATP) and cardiac (200 µM Ca²⁺) RyRs, cytosolic RR induced a predominant subconductance at a positive but not negative holding potential. Lumenal RR induced a major subconductance in cardiac RyR at negative but not positive holding potentials and several subconductances in skeletal RyR. The RR-related subconductances of cardiac RyR showed a nonlinear voltage dependence, and more than one RR molecule appeared to be involved in their formation. Cytosolic and lumenal RR also induced subconductances in Ca²⁺-conducting skeletal and cardiac RyRs recorded at 0 mV holding potential. These results suggest that RR inhibits RyRs and induces subconductances by binding to cytosolic and lumenal sites of skeletal and cardiac RyRs.

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